Proteomic approaches to protein fatty acylation Protein lipidation is a fundamentally important cellular process that functions in protein folding, protein targeting, membrane interaction and protein/protein interactions. It plays significant roles in cancer and neurodegenerative disease though our understanding of the exact nature of these roles is relatively poor. Discovery of new protein lipidation targets will potentially impact other diseases. Unfortunately, shotgun proteomics workflows that simplify proteins to a collection of peptides tend not to detect lipidated peptides, probably because the protocols employed are optimized to the most easily recovered soluble peptides (proteotypic). Addressing the bias against protein lipidation detection in proteomics requires novel innovations in separations technologies for bottom-up approaches and specialized top-down or middle-down approaches when targeting intact lipidated proteins. Specific aim one will focus on efficient digestion of lipidated proteins and subsequent recovery of lipidated peptides via enrichment with novel HILIC chromatography protocols. We will also innovate modified reverse-phase protocols for recovery of long- chain acylated peptides and tandem mass spectrometry. The overall goal will be a robust two-dimensional separation technology for proteome-wide detection and characterization of lipidated peptides. Specific aim two will focus development of top-down mass spectrometry technologies for characterization of lipidated proteins. This aim includes MSMSMS strategies as well as middle-down strategies to target robust localization of lipidation sites. The overall goal will be a robust mass spectrometry /protein chemistry technology for top-down analysis of protein lipidation across the proteome. PUBLIC HEALTH RELEVANCE: The diversity of proteins coded in the human genome perform the majority of cellular functions that allow human beings to thrive. Some proteins are modified to make them greasy (fatty acylation or lipidation) thereby modulating their function desirably, or in diseases including some cancers and possibly Alzheimer's Disease undesirably. We propose to develop new proteomics technologies to accurately describe and measure this important protein modification that has to date been poorly detected.